Isotope Dilution Approach Research Articles

Quantification of PFAS in rice and maize: Validation of a UHPLC-HRMS/MS isotopic dilution approach in support to food safety

In the present work, an analytical method for the quantification of per and poly fluoroalkyl substances (PFAS) in rice and maize has been developed and then validated with a metrological approach. PFAS are a group of human-made chemicals used in a variety of industries and consumer products for their water- and grease-resistant properties. Studies have shown that PFAS can contaminate soil and water, and there is concern about their bioaccumulation in edible plants, fruits, and cereals. The presence of PFAS has been identified in rice and other food products, including maize, as indicated by studies and scientific literature. This is particularly alarming since some PFAS have been associated with adverse health effects and rice and maize account for over 20% of the annual food intake worldwide. Despite this evidence, the regulation currently in place is not covering cereal matrices and limits of quantification for matrices encompassed by the current legislation are defined for a small group of PFAS. In this study an UHPLC-HRMS/MS based method was validated, obtaining a LOQ (Limit Of Quantification) ranging between 2 ng/kg and 32 ng/kg and robustness in line with EU guidelines and recommendation for PFAS in food. Additionally, a metrological approach was employed to estimate the uncertainty budget, utilizing modeling and experimental methods, and comparing the outcomes, aiming to characterize with high accuracy PFAS in rice and maize and support control bodies to assess contamination in suspected areas. A comparison of uncertainty of different approaches was conducted after applying the method to 30 real samples.

Development of an SI-traceable N-terminal pro-B-type natriuretic peptide certified reference material using structure-based impurity-corrected isotope dilution mass spectrometry approaches.

N-Terminal pro-B-type natriuretic peptide (NT-proBNP) is a pivotal biomarker for the diagnosis and prognosis of heart failure (HF). However, no SI-traceable certified reference material (CRM) or reference measurement procedure (RMP) is available for this biomarker, and so clinical testing results obtained in different laboratories cannot be traced to a higher-order standard, leading to incomparable measurements. Protein hydrolysis and protein cleavage isotope dilution mass spectrometry (AAA-IDMS and PepA-IDMS) were used to develop a CRM. Structurally related impurities were identified by high-resolution mass spectrometry. The quantitative AAA-IDMS results were corrected according to the amino acid compositions of the impurities. Using PepA-IDMS, two peptides from the proteolyzed product were confirmed as signature peptides. To obtain traceable and accurate results, the signature peptides were quantified using impurity-corrected AAA-IDMS. The candidate NT-proBNP solution was denatured and enzymatically digested using the Glu-C endoproteinase. The released signature peptides were measured using an isotopic dilution approach. The homogeneity and stability of the candidate CRM were characterized, and their uncertainties were combined with the value assignment process. The developed CRM can be considered a unique SI-traceable NT-proBNP reference material and is expected to be used as a primary calibrator for matrix NT-proBNP CRM development.

Probe Electrospray Ionization Tandem Mass Spectrometry for the Detection and Quantification of Benzodiazepines.

Legally prescribed benzodiazepines (BZDs) and designer BZDs are widely misused and must be determined in multiple contexts (eg, overdose, drug-facilitated sexual assaults, or driving under the influence of drugs). This study aimed to develop a method for measuring serum BZD levels using probe electrospray ionization (PESI) mass spectrometry and an isotope dilution approach. A tandem mass spectrometer equipped with a probe electrospray ionization source in multiple reaction monitoring mode was used. Isotope dilution was applied for quantification using a deuterated internal standard at a fixed concentration for alprazolam, bromazepam, diazepam, nordiazepam, oxazepam, temazepam, zolpidem, and zopiclone. This method included designer BZDs: clonazolam, deschloroetizolam, diclazepam, etizolam, flualprazolam, flubromazepam, flubromazolam, meclonazepam, nifoxipam, and pyrazolam. Sample preparation was done by mixing 10 µL of serum with 500 µL of an ethanol/ammonium formate 0.01 mol/L buffer. Complete validation was performed, and the method was compared with liquid chromatography coupled with mass spectrometry (LC-MS/MS) and immunoassays (IC) by analyzing 40 real samples. The analysis time for identification and quantification of the 18 molecules was 2.5 minutes. This method was fully validated, and the limits of quantification varied from 5 to 50 mcg/L depending on the molecule. In the 40 real samples, 100% of molecules (n = 89) were detected by both LC-MS/MS and PESI-MS/MS, and regression analysis showed excellent agreement between the 2 methods (r 2 = 0.98). On IC, bromazepam and zolpidem were not detected in 2 and 1 cases, respectively. PESI-MS/MS allows serum BZD detection and measurement. Given the isotope dilution approach, a calibration curve was not required, and its performance was similar to that of LC-MS/MS, and its specificity was higher than that of IC.

Development of an Online Isotope Dilution CE/ICP-MS Method for the Quantification of Sulfur in Biological Compounds.

We report an analytical methodology for the quantification of sulfur in biological molecules via a species-unspecific postcolumn isotope dilution (online ID) approach using capillary electrophoresis (CE) coupled online with inductively coupled plasma-mass spectrometry (online ID CE/ICP-MS). The method was optimized using a mixture of standard compounds including sulfate, methionine, cysteine, cystine, and albumin, yielding compound recoveries between 98 and 105%. The quantity of sulfur is further converted to the quantity of the compounds owing to the prior knowledge of the sulfur content in the molecules. The limit of detection and limit of quantification of sulfur in the compounds were 1.3-2.6 and 4.1-8.4 mg L-1, respectively, with a correlation coefficient of 0.99 within the concentration range of sulfur of 5-100 mg L-1. The capability of the method was extended to quantify albumin in its native matrix (i.e., in serum) using experimentally prepared serum spiked with a pure albumin standard for validation. The relative expanded uncertainty of the method for the quantification of albumin was 6.7% (k = 2). Finally, we tested the applicability of the method on real samples by the analysis of albumin in bovine and human sera. For automated data assessment, a software application (IsoCor)─which was developed by us in a previous work─was developed further for handling of online ID data. The method has several improvements compared to previously published setups: (i) reduced adsorption of proteins onto the capillary wall owing to a special capillary-coating procedure, (ii) baseline separation of the compounds in less than 30 min via CE, (iii) quantification of several sulfur species within one run by means of the online setup, (iv) SI traceability of the quantification results through online ID, and (v) facilitated data processing of the transient signals using the IsoCor application. Our method can be used as an accurate approach for quantification of proteins and other biological molecules via sulfur analysis in complex matrices for various fields, such as environmental, biological, and pharmaceutical studies as well as clinical diagnosis.

Production and certification of BOTS-1: bovine muscle-certified reference material for incurred veterinary drug residues.

A freeze-dried bovine muscle-certified reference material (CRM), known as BOTS-1 (DOI: https://doi.org/10.4224/crm.2018.bots-1 ), containing incurred residues of commonly used veterinary drugs was produced and certified for the mass fraction of eight veterinary drug residues. Value assignment was carried out using liquid chromatography tandem mass spectrometry (LC-MS/MS) methods in conjunction with isotope dilution and standard addition approaches involving stable isotope internal standards. Data from the National Research Council of Canada (NRC), Canadian Food Inspection Agency (CFIA), United States Department of Agriculture (USDA), and the Federal Office of Consumer Protection and Food Safety in Germany (BVL) were used for value assignment. Results for two drug residues were also obtained through an international inter-laboratory comparison CCQM-K141/P178 organized under the auspices of the International Bureau of Weights and Measures (BIPM). Quantitative NMR (1H-qNMR) was used to characterize primary standards of all veterinary drugs certified. The certified mass fractions of the veterinary drug residues were 490 ± 100µg/kg for chlorpromazine, 44 ± 4.4µg/kg for ciprofloxacin, 3.3 ± 1.4µg/kg for clenbuterol, 9.5 ± 0.8µg/kg for dexamethasone, 57 ± 4.8µg/kg for enrofloxacin, 3.0 ± 0.4µg/kg for meloxicam, 12.4 ± 1.2µg/kg for ractopamine, and 2290 ± 120µg/kg for sulfadiazine with expanded uncertainties quoted (95% confidence) which include the effects due to between-bottle inhomogeneity, instability during long-term storage and transportation, and characterization.

Nutrient interactions and salinity effects on plant uptake of phosphorus from waste-based fertilisers

Many organically managed farms in Europe have low levels of soil phosphorus (P). Arable farms that rely strongly on biological nitrogen fixation (BNF) have been shown to have rather low outputs and a tendency to deplete soil P and potassium (K) compared with arable farms that have a lower reliance on BNF and higher external inputs. Therefore, research focusing on providing a balanced input of nitrogen (N), P, K and sulphur (S) from alternative sources is of interest to organically managed farms in Europe. The aim of this study was to quantify P availability from different organic wastes applied alone or in combination to improve the mixtures’ N:P:K:S ratio. P availability was measured by P uptake and recovery in ryegrass grown in pots. The isotope dilution approach was used in which a non-labelled fertiliser is added to a soil that has been pre-incubated and equilibrated with labelled 33P. The P recovery of the different organic wastes varied significantly (10–20 %). Manure and anaerobically digested manure mixed with ash from straw had the lowest P recovery. All the organic waste treatments had higher plant growth and P uptake compared with the negative control, but none of them reached the values observed after application of mineral P. Mixing digested manure with ash increased soil pH at the end of the experiment, which may explain the lower P availability. The highest P recovery was found in digested products, either manure alone or mixed with municipal waste or the industrial waste product Fertigro®. However, the mixture of digested manure and Fertigro® led to lower dry matter production, whereas Fertigro® used alone resulted in high leaf P concentrations but depressed shoot and root growth, presumably due to salinity effects and a decrease in soil pH. Anaerobic digestion increased the availability of P, which may be explained by the lower immobilisation potential of the remaining organic matter in the digestate. This study highlights the potential challenges when attempting to improve the N:P:K:S ratios of waste-based fertilisers through mixing due to material interactions. However, such effects are likely to be overexpressed in pot trials that have a limited soil volume. Field trials are therefore needed to quantify such effects in practice.

Ultrafast measurement of drugs using Probe Electrospray Ionization Mass Spectrometry (PESI-MS) and isotope dilution: An example with metformin

Probe Electrospray Ionization (PESI) is a miniaturized electrospray (ESI) with a capillary of the nanoelectrospray converging on a solid needle with a tip of several hundred nanometers. This probe was developed to be coupled directly to a mass spectrometer (MS) without the need for chromatographic separation, allowing ultrafast analysis with minimal sample preparation. Metformin is the first-line treatment for type 2 diabetes. Lactic acidosis (LA) is a frequent complication that can be either induced by or associated with elevated metformin plasma concentrations. Therefore, a rapid measurement plays an important role in helping clinicians to diagnose overexposure to metformin. In the present study, our objective was to develop a PESI-MS method for an ultra-rapid determination of metformin in plasma by using an isotope dilution approach. Analysis was carried out with a PESI source installed on a triple quadrupole MS (LCMS 8060®, Shimadzu). A multiple reaction monitoring mode (MRM) with 2 transitions for metformin and metformin-D6 (internal standard) was used. To be able to apply an isotope dilution protocol, the main MS parameters were optimized to ensure that the m/z 130.15 > 71.10 (metformin) and m/z 135.95 > 77.15 (metformin-D6) transitions exhibited the same intensity. The method was fully validated with 6 concentration levels (0.5 to 50 mg/L). The matrix effect was evaluated for each level, and the specificity was tested by analysing 6 plasmas spiked with a mix of 44 potential co-medications. Finally, the performance was compared with 2 classical LC-MS/MS and LC-DAD methods used in external labs, by analysing samples from 29 patients. The sample preparation consisted in mixing 10 μL of plasma with 1000 μL ethanol/ammonium formate buffer including metformin-D6 at a fixed concentration of 5 mg/L. Total run time was 0.31 min. The limit of detection and quantification was fixed at 0.5 mg/L. Isotope dilution gave satisfactory results in accuracy and precision for each concentration level (min-max: −12.1–15.8% and 1.0–7.1%, respectively). Thanks to the internal standard the matrix effect was fully corrected (bias less than 1%). The specificity study also reported satisfactory results. Finally, in the representative group of 29 patients (55% with a concentration <5 mg/L, 38% with a concentration >5 mg/L and 7% not detected), we observed almost identical results when comparing LC-DAD and LC-MS/MS to PESI-MS (r2 > 0.99). The PESI-MS approach needs no sample preparation. Combined with a isotope dilution protocol, that requires no calibration curve building, offers the best possibility to design specific, sensitive, accurate and ultra-fast solutions. Our study presents a method which allows an accurate determination of metformin in about 30 seconds that will be helpful in a core lab when rapid diagnosis of LA is needed.

Ultrafast Measurement of Metformin in the Clinical Setting Using Probe Electrospray Ionization Mass Spectrometry.

Metformin (MtF) is a treatment used for type 2 diabetes. Lactic acidosis (LA) is a frequent complication that can be either induced by or associated with elevated MtF plasma concentrations. When coupled with a mass spectrometry (MS) system, the probe electrospray ionization (PESI) method allows direct and rapid analysis of different types of matrices without pretreatment. In this study, we developed a PESI-MS method for the determination of MtF in plasma. We used a tandem mass spectrometer equipped with a PESI source in the reaction monitoring mode for the quantitation of MtF. MtF-d6 was chosen as the internal standard (IS), following an isotope dilution (ID) approach. The method was fully validated with six concentration levels (0.5-50 mg/L). The matrix effect was evaluated for each level, and the specificity was tested with a mix of potential co-medications. Using patient samples, the performance was compared with two classical LC-MS-MS and LC-diode array detector (DAD) methods used in external labs. Sample preparation consisted in mixing 10 µL plasma in 1,000 µL ethanol/ammonium formate buffer including MtF-d6 at a fixed concentration of 5 mg/L. The total run time was 0.31 min. ID gave satisfactory results of accuracy and precision (min-max: -12.1 to 15.8% and 1.0-17.1%, respectively). The matrix effect was fully corrected by the internal standard (bias < 1%). The specificity study also reported satisfactory results. Finally, in a representative group of 29 patients (55% with a concentration <5 mg/L, 38% with a concentration >5 mg/L and 7% not detected), we observed almost identical results when comparing LC-DAD and LC-MS-MS to PESI-MS (r2 > 0.99). We propose a specific, sensitive, accurate and ultrafast solution for the measurement of MtF in patient plasma, with no sample preparation or calibration curve building. This could be helpful in a core lab when rapid diagnosis of LA is needed.

Antagonistic Interaction of Selenium and Cadmium in Human Hepatic Cells Through Selenoproteins.

Cadmium (Cd) is a highly toxic heavy metal for humans and animals, which is associated with acute hepatotoxicity. Selenium (Se) confers protection against Cd-induced toxicity in cells, diminishing the levels of ROS and increasing the activity of antioxidant selenoproteins such as glutathione peroxidase (GPx). The aim of this study was to evaluate the antagonistic effect of selenomethionine (SeMet) against Cd toxicity in HepG2 cells, through the modulation of selenoproteins. To this end, the cells were cultured in the presence of 100 µM SeMet and 5 μM, 15 µM, and 25 µM CdCl2 and a combination of both species for 24 h. At the end of the experiment, cell viability was determined by MTT assay. The total metal content of Cd and Se was analyzed by triple-quadrupole inductively coupled plasma–mass spectrometry (ICP-QqQ-MS). To quantify the concentration of three selenoproteins [GPx, selenoprotein P (SELENOP), and selenoalbumin (SeAlb)] and selenometabolites, an analytical methodology based on column switching and a species-unspecific isotopic dilution approach using two-dimensional size exclusion and affinity chromatography coupled to ICP-QqQ-MS was applied. The co-exposure of SeMet and Cd in HepG2 cells enhanced the cell viability and diminished the Cd accumulation in cells. Se supplementation increased the levels of selenometabolites, GPx, SELENOP, and SeAlb; however, the presence of Cd resulted in a significant diminution of selenometabolites and SELENOP. These results suggested that SeMet may affect the accumulation of Cd in cells, as well as the suppression of selenoprotein synthesis induced by Cd.

Development of a novel approach for the second-tier estimation of homocysteine in dried blood spot using tandem mass spectrometry

Tandem mass spectrometry (TMS) -based screening for homocystinuria in the routine newborn screening (NBS) primarily relies on the elevated levels of methionine (Met) in the dried blood spot (DBS) of affected neonates. However, due to the lack of required specificity of Met as a marker for homocystinuria, an additional re-sampling and follow-up analytical testing for a definitive diagnosis, emphasis us to develop second-tier testing for a more specific marker, that is homocysteine (HCY) in the same residual blood spot without any new sample. Objective: The objective of the study was to develop a novel approach for the second-tier estimation of total HCY in residual blood spot using TMS. Materials and Methods: Retrospective analysis of DBS from all neonates with deranged levels of Met and Met to phenylalanine in primary NBS was included in the study. The study duration was for a period of 24 months (November 2015-October 2017). Heel prick samples were collected within 24–72 h of birth and after the first feed. Further, the samples with abnormal Met was analyzed for total HCY by isotopic dilution approach using multiple reaction monitoring modes for mass transition m/z 136?90 for HCY, m/z 140?94 for d4-homocysteine, and m/z 269.2?134.4 for d8-HCY. Results: The newly developed methodology demonstrated a sensitive method for the quantification of total HCY over a linear range of 2 ?mol/L–100 ?mol/L. Assay validation parameters such as intraday and interday assay precision, accuracy, and recovery were also within an acceptable range. Conclusion: In the present study, an in-house tandem mass spectrometric-based assay for the quantification of total HCY was developed which can be employed as an important strategy for the differential diagnosis of homocystinuria in neonates.

A New Approach for Second-tier Analysis of Methylmalonic Acid in Dried Blood Spots using Liquid Chromatography Tandem Mass Spectrometry

Introduction: Inactivity or diminished activity of an enzyme Methylmalonyl-CoA mutase (a Cobalamin dependent) enzyme causes inborn error of metabolism named Methylmalonic Acidemia/Aciduria (MMA). Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) based method for the diagnosis of MMA in Newborn Screening (NBS), is often challenging due to the nonspecificity of propionylcarnitine (C3), a primary marker in routine NBS. Aim: To develop a Flow Injection Analysis (FIA) method for the second-tier estimation of Methylmalonic acid in the Dried Blood Spots (DBS) of primary NBS. Materials and Methods: A retrospective NBS study was conducted for a period of two years i.e. (November 2015 to November 2017) at the Paediatrics Research and Genetic Lab of Maulana Azad Medical College associated Lok Nayak Hospital, a Tertiary Care Centre in New Delhi, India. DBS samples were collected by heel- prick method and a second tier detection, quantification of methylmalonic acid was performed by LC-MS/MS on all samples with abnormal C3 levels in primary NBS. Multiple Reaction Monitoring (MRM) mode at m/z 117→73 for MMA and m/z 120→75 for MMA(IS) and isotopic dilution approach was followed for quantification. Results: Intra-assay and inter-assay precision and accuracy was determined at two different levels of MMA (LQC≅2.0 μmol/L and HQC≅10.0 μmol/L), respectively. The Coefficient of Variation (%) for intraday precision ranged between 5.27% to 8.9%. Similarly, for interday it ranged from 4.99% to 9.93%. The average accuracy (%) also falls within (105.4% and 106.1%) for interday and (105.9% and 106.7%) for intraday assay. Stability for samples during storage at different temperature i.e., (fresh, 2-8°C and -20°C) showed long term stability at -20°C storage. The assay was linear over a calibration range of (0.5 to 20.00 μmol/L). Conclusion: The outcome of the present data offers the confidence and reliability in the possible utility of this method for the definitive diagnosis and follows-up of MMA patients.

A method of assessing essential amino acid availability from microbial and ruminally undegraded protein in lactating dairy cows

The objective of this study was to extend a stable isotope-based assessment of AA absorption from rumen-degradable protein (RDP) sources to include determination of essential AA (EAA) availability from microbial protein (MCP). To demonstrate the technique, a study using a 2 × 2 factorial arrangement of treatments applied in a repeated 4 × 4 Latin square design was undertaken. Factors were high and low rumen-degradable protein and high and low starch. Twelve lactating cows were blocked into 3 groups according to days in milk and randomly assigned to the 4 treatment sequences. Each period was 14 d in length with 10 d of adaption followed by 4 d of ruminal infusions of 15N-labeled ammonium sulfate. On the last day of each period, a 13C-labeled AA mixture was infused into the jugular vein over a 6-h period to assess total AA entry. Rumen, blood, urine, and milk samples were collected during the infusions. Ruminal bacteria and blood samples were assessed for AA enrichment. Total plasma AA absorption rates were derived for 6 EAA from plasma 13C AA enrichment. Absorption of 6 EAA from MCP was calculated from total AA absorption based on 15N enrichment in blood and rumen bacteria. Essential AA absorption rates from total protein, MCP, and rumen-undegradable protein were derived with standard errors of the mean of 6, 14, and 14%, respectively. An average of 45% of absorbed EAA were from MCP, which varied among 6 EAA and was interactively affected by starch and RDP in diets. Microbial AA availability measured by isotope dilution method increased with the high RDP diets and was unaffected by starch level, except for Met, which decreased with high starch. Microbial protein outflow, estimated from urinary purine derivatives, increased with RDP and was not significantly affected by starch. This was consistent with measurements from the isotope dilution method. Total AA absorption rates measured from isotope dilution were similar to estimates from CNCPS (v. 6.55), but a lower proportion of absorbed AA was derived from MCP for the former method. Compared with the isotope and CNCPS estimates, the Fleming model underestimated microbial EAA and total EAA availability. An average of 58% of the absorbed EAA was converted into milk, which varied among individual AA and was interactively affected by starch and RDP in diets. The isotope dilution approach is advantageous because it provides estimates of EAA availability for individual EAA from rumen-undegradable protein and MCP directly with fewer errors of measurement than can be achieved with intestinal disappearance methods.

Insights into Primary Ion Exchange between Ion-Selective Membranes and Solution. From Altering Natural Isotope Ratios to Isotope Dilution Inductively Coupled Plasma Mass Spectrometry Studies.

Although ion-selective electrodes have been routinely used for decades now, there are still gaps in experimental evidence regarding how these sensors operate. This especially applies to the exchange of primary ions occurring for systems already containing analyte ions from the pretreatment step. Herein, for the first time, we present an insight into this process looking at the effect of altered ratios of naturally occurring analyte isotopes and achieving isotopic equilibrium. Benefiting from the same chemical properties of all isotopes of analyte ions and spatial resolution offered by laser ablation and inductively coupled plasma mass spectrometry, obtaining insights into primary ion diffusion in the preconditioned membrane is possible. For systems that have reached isotopic equilibrium in the membrane through ion exchange and between the membrane phase and the sample, quantification of primary ions in the membrane is possible using an isotope dilution approach for a heterogeneous system (membrane–liquid sample). Experimental results obtained for silver-selective membrane show that the primary ion diffusion coefficient in the preconditioned membrane is close to (6 ± 1) × 10–9 cm2/s, being somewhat lower compared to the previously reported values for other cations. Diffusion of ions in the membrane is the rate limiting step in achieving isotopic exchange equilibrium between the ion-selective membrane phase and sample solution. On the contrary to previous reports, quantification of silver present in the membrane clearly shows that contact of the membrane with silver nitrate solution of concentration 10–3 M leads to pronounced accumulation of silver ions in the membrane, reaching almost 150% of ion exchanger amount. The magnitude of this effect increases for higher concentration of the electrolyte in the solution.

Application of a streamlined LC-MS/MS methodology for the determination of atropine and scopolamine in cereals from Asian and African countries

ABSTRACT Tropane alkaloids are toxic secondary metabolites produced by a wide variety of plants that can be present in edible materials or animal feed. Several human poisoning cases through consumption of cereals were reported over the last years and highlighted the need for reliable and robust analytical methodologies for safety control. To rationalize analyses in high-throughput laboratory environments dealing with shorter and shorter turn-around-around time, the scope of our multi mycotoxins method was extended to the analysis of two regulated tropane alkaloids, namely atropine and scopolamine. Extraction procedure is based on the QuEChERS (Quick, Easy, Cheap, Efficient, Rugged, and Safe) approach followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) detection. Quantification is performed by the isotopic dilution approach using labelled isotopomers as internal standard. The procedure was validated at two fortification levels (0.5 µg/kg and 10 µg/kg) on different cereal-based products according to the European SANTE/12682/2019 document and performance parameters such as precision (RSD(r) ≤ 6%, RSD(iR) ≤ 6%) and recovery (82–114%) fulfilled its requirements. The limit of quantification (0.5 µg/kg) is low enough to ensure compliance with existing regulations. The method was further applied on 95 cereals and cereal-based products collected from Asian and African countries. All samples were found free of the two targeted TAs, with the exception of a rice-based product in which both atropine and scopolamine were quantified at 9.6 µg/kg and 2.6 µg/kg, respectively. A total of 29 cereals samples, shown to be free of both atropine and scopolamine were also analysed for mycotoxins. Aflatoxins, fumonisins, and deoxynivalenol were sporadically detected at levels below the maximum levels defined by the European Union legislation often considered as the most stringent regulation.

Improved fully automated method for the determination of medium to highly polar pesticides in surface and groundwater and application in two distinct agriculture-impacted areas.

Water is an essential resource for all living organisms. The continuous and increasing use of pesticides in agricultural and urban activities results in the pollution of water resources and represents an environmental risk. To control and reduce pesticide pollution, reliable multi-residue methods for the detection of these compounds in water are needed. In this context, the present work aimed at providing an analytical method for the simultaneous determination of trace levels of 51 target pesticides in water and applying it to the investigation of the target pesticides in two agriculture-impacted areas of interest. The method developed, based on an isotopic dilution approach and on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry, is fast, simple, and to a large extent automated, and allows the analysis of most of the target compounds in compliance with European regulations. Application of the method to the analysis of selected water samples collected at the lowest stretches of the two largest river basins of Catalonia (NE Spain), Llobregat and Ter, revealed the presence of a wide suite of pesticides in the Llobregat basin, some of them at concentrations above the water quality standards (irgarol and dichlorvos) or the acceptable method detection limits (methiocarb, imidacloprid, and thiacloprid), and much cleaner waters in the Ter River basin. Risk assessment of the pesticide concentrations measured in the Llobregat River indicated high risk due to the presence of irgarol, dichlorvos, methiocarb, azinphos ethyl, imidacloprid, and diflufenican (hazard quotient (HQ) values>10), and moderate potential risk in the Ter River, associated to the occurrence of bentazone and irgarol (HQ > 1).

Paper spray tandem mass spectrometry: A rapid approach for the assay of parabens in cosmetics and drugs.

A fast methodology for the assay of parabens in drug and cosmetic preparations has been presented. The procedure developed is based on paper spray tandem mass spectrometry and isotope dilution approach. For each investigated paraben, the corresponding labeled standard has been used in order to improve the accuracy and reproducibility of the analyses. The MS experiments have been performed under MRM conditions, monitoring the transitions [M-H]- → m/z 92 and [M-H]- → m/z 98, respectively, for each analyte and the corresponding labeled internal standard. The quantitative assay has been performed using a calibration curve built from 2 to 15 mg/L. The method accuracy, in all case near 100%, was evaluated using fortified samples at two concentration levels, which are representative of the lower and the higher portion of calibration curve. The good values of LOQ, LOD, and reproducibility confirm the consistency of the developed approach.

Sample pre-treatment and analytical methodology for the simultaneous determination of pharmaceuticals and personal care products in sewage sludge

This work describes the design, optimization and validation of an analytical method for the simultaneous determination of 14 pharmaceuticals and personal care products (PPCPs) in sewage sludge. A thorough optimization of the sample pre-treatment was carried out. As a result, microwave-assisted extraction (MAE) was combined with an in-situ clean-up stage and a filtration step. A combination of MilliQ® water/MeOH 95:5 (v/v) adjusted to pH 9 turned out to be the optimal solvent mixture for extraction. The instrumental part of the method presents a significant novelty based on a fully automated sample preparation for the analysis of PPCPs. It consisted of a direct immersion solid phase microextraction followed by on-fiber derivatization, online coupled to gas chromatography - mass spectrometry (DI-SPME-On-fiber derivatization - GC-MS). An isotope dilution approach was used for quantifying, which conferred high reliability to the method. This methodology was validated for 10 compounds with good analytical performance, limit of detection below 20 ng g−1 and absolute recovery in the range of 30–70% for most of the compounds. It supposes an ecological analytical alternative for many routine analysis laboratories around the world. The developed method was applied to different real samples generated in both a pilot-scale thermal hydrolysis treatment plant and an anaerobic digester operated in mesophilic conditions. Salicylic acid and naproxen were found at concentrations above 1000 ng g−1.

Quantification of light polycyclic aromatic hydrocarbons in seafood samples using on-line dynamic headspace extraction, thermodesorption, gas chromatography tandem mass spectrometry, based on an isotope dilution approach

The aim of our work was to develop an analytical strategy to quantify naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene and anthracene in fish products by on-line dynamic headspace extraction, followed by thermodesorption injection and gas chromatography analysis coupled with tandem mass spectrometry using electron ionization mode (DHS-TD-GC-EI-MS/MS). The developed protocol used 1 g of freeze-dried or oil sample supplemented with perdeuterated light PAHs. The sample was heated at [90 –100 °C], the headspace of the sample was swept by nitrogen and the trapping of the PAHs was carried out on a Tenax-type adsorbent placed at 25 °C. Analytes were thermodesorbed at 300 °C from the dried adsorbant and then cryofocused on a cooled injection system (CIS) at −25 °C before injection (12 °C s−1 up to 300 °C). The chromatographic separation of PAHs was carried out on a 5-MS type column (30 m × 0.25 mm, 0.25 μm) and the acquisition of the signals was performed in SRM following the transitions, involving the loss of one or two hydrogen atoms from the molecular ion. In view of the principle of extraction, the calibration curve was performed on a representative matrix or using the standard addition method. Quantification limits were determined between 0.01 and 0.6 ng g−1 of matrix from the method blank results. The method was validated by a series of multi-level supplemented matrix assays and by the analysis of a reference material from an inter-laboratory test (mussels, IAEA-432). The average of the expanded measurement uncertainty was from 9 to 44% for the four lightest PAHs, except for fluorene when the sample incubation was set at 90 °C. Occurrence measurements were performed on almost two hundred samples of molluscs, echinoderms and fish. The results have shown a quantification frequency greater than 66% for naphthalene and fluorene, at concentrations below a few ng g−1 of dry matter of fishery products. With this methodology, the light PAHs occurrence can now be measured in a wider range of foodstuffs in order to better characterize their contamination trends and the associated risk simultaneously.

A reliable LC-MS/MS-based method for trace level determination of 50 medium to highly polar pesticide residues in sediments and ecological risk assessment.

The occurrence of polar pesticides in sediments has not been extensively investigated because of their relatively poor hydrophobicity and apparently less persistence in the environment. However, their continuous release into the aquatic systems calls for the evaluation of their potential accumulation in sediments and the role of this matrix as a potential source of these compounds. Considering this, a method based on pressurized liquid extraction (PLE), extract clean-up by solid phase extraction (SPE), and analyte determination by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed and validated to analyze 50 relevant (frequently used and/or regulated or found in water) medium to highly polar pesticides in sediments. The method showed good performance regarding accuracy (relative recoveries between 76 and 124%), precision (relative standard deviation values < 20%), sensitivity (LODs in the low nanogram per gram for most compounds), linearity (coefficients of determination > 0.99), and matrix effects (negligible for all analytes). The use of an isotope dilution approach for quantification ensures result reliability. As a part of the validation process, the method was applied to the analysis of the target pesticides in sediments from the Llobregat River (NE Spain) showing the presence of five of them, namely, terbutryn, dichlorvos, terbuthylazine, diazinon, and irgarol. All 5 pesticides, due to both the concentrations found and their physical-chemical characteristics, demonstrate high potential for bioaccumulation and risk to aquatic organisms. Additional multi-disciplinary studies that investigate pesticide occurrence in different aquatic compartments and evaluate the potential risks for aquatic ecosystems are required to assess the environmental impact and significance of the presence of pesticides in sediments. Graphical Abstract.

A stable isotope dilution approach to analyze ferulic acid oligomers in plant cell walls using liquid chromatography-tandem mass spectrometry.

Diferulic (DFA) and triferulic acids (TriFA) acylate and cross-link plant cell wall polysaccharides, thereby being important structural elements within the cell wall, also affecting physicochemical properties of the isolated polysaccharides. Due to the large number of potential regio- and configurational isomers and due to the fact that oligoferulic acids are not commercially available as standard compounds, analysis of oligoferulic acids after alkaline hydrolysis is challenging. Eighteen di- and triferulic acids were synthesized both non-labeled as well as 13C-labeled. By using these standard compounds, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) (electrospray ionization, negative mode)-based stable isotope dilution approach was developed, fully validated and applied to plant materials. Whereas this stable isotope dilution approach is most useful to analyze plant materials with complex matrices (especially lignified tissues), less complicated matrices may not require this approach. Therefore, an alternative LC-MS/MS-based method that is based on using a single internal standard compound only was developed, too, validated, and compared to the stable isotope dilution approach. Although the stable isotope dilution approach appears to be superior, plant samples with simple matrices can also be screened by using the single internal standard method developed here.